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Previous Article | Next Article 
J Virol, May 1998, p. 4448-4453, Vol. 72, No. 5
Division of Molecular Virology, Baylor
College of Medicine, Houston, Texas
Received 14 October 1997/Accepted 3 February 1998
The human cdc2-related kinase PITALRE is the catalytic component of
TAK, the Tat-associated kinase. Previously, we have proposed that TAK
is a cellular factor that mediates Tat transactivation function. Here
we demonstrate that transient overexpression of PITALRE specifically
squelches Tat-1 activation of both a transfected and an
integrated human immunodeficiency virus type 1 (HIV-1) long terminal
repeat (LTR), suggesting that PITALRE mediates Tat function as a
multiprotein complex. A catalytic mutant of PITALRE, D167N, was
found to be more efficient than wild-type PITALRE in squelching Tat
transactivation. Neither wild-type PITALRE nor D167N was able to
squelch transactivation of the human T-cell leukemia type 1 LTR by the
Tax protein. Additionally, we show that artificial targeting of PITALRE
to a nascent RNA element, in the absence of Tat, activated HIV-1 LTR
expression. These results indicate that a PITALRE-containing complex
mediates transactivation by Tat and suggest that Tat proteins function
by localizing such a PITALRE-containing complex to the site of
the transcribing provirus.
The proviral genome of human
immunodeficiency virus type 1 (HIV-1) and HIV-2 is transcribed by
the host cell RNA polymerase II (RNAPII) complex. The viral RNAs are
subject to complex patterns of splicing that yield both messenger and
genomic viral RNA products. Both HIV RNA synthesis and splicing, while
dependent on host functions, are subject to regulation by viral
functions. Autoregulation of HIV transcription is primarily mediated by
the HIV Tat protein (2, 35), and Tat function is essential
for efficient virus replication (7, 9). Tat interacts
directly with the nascently transcribed transactivation response region
(TAR) RNA structure at the 5' end of all viral transcripts, and this
interaction is a requisite for maximal Tat-mediated upregulation of
viral transcription (reviewed in reference 16).
Genetic experiments have identified an activation domain within Tat
that can function independently of Tat's RNA-binding domain to
activate transcription (17, 33, 34, 36).
In the absence of Tat, the HIV promoter specifies primarily abortive
transcription complexes, whereas in the presence of Tat, the quantity
of full-length transcripts is greatly increased (18, 20,
26). These observations have suggested that the HIV-1 promoter
assembles primarily transcription complexes which are subject to
abortive elongation and that Tat acts by mediating an alteration of the
initiated complex such that the processivity of the transcription
complex is enhanced. It thus appears likely that Tat effects a discrete
step during the RNAPII transcription cycle.
Genetic and biochemical data indicate the existence of one or more
cellular cofactors that mediate the transcriptional effect of Tat
proteins. It has been observed that the activation domains of the
lentiviral Tat proteins of HIV and equine infectious anemia virus
can squelch Tat transactivation, suggesting that a limiting cellular target exists (3, 22). Such a cofactor should
interact specifically with the functional activation domains of Tat
proteins and should enable Tat proteins to activate elongation of
transcription. Studies in vitro with fractionated systems suggest that
Tat-mediated transcription requires a cellular function which is not
conferred by general transcription factors (37, 45). Based
on biochemical observations, a number of cofactors that could
constitute the cellular interface to Tat function have been proposed,
including cellular general transcription factor TFIIH (11,
31), Tat-SF1 (46), and Tat-associated kinase
(TAK) (14, 15, 42).
As Tat proteins act to stabilize RNAPII elongation, the
biochemical events associated with RNAPII elongation have
served as the basis for mechanistic analyses of Tat. Of
significance in this regard are the findings that (i) elongation of
RNAPII transcription can be specifically inhibited by the
nucleoside analog
5,6-dichloro-1- Recent experiments in this laboratory (41) indicate that the
42-kDa phosphorylated protein which copurifies with TAK activity is a previously identified cdc2-related protein kinase, PITALRE (13). PITALRE is ubiquitously expressed in human tissues,
has no apparent cell cycle regulation, and associates with
several additional cellular proteins in vivo (12, 13).
Antibodies to PITALRE deplete HeLa cell nuclear extracts of
TAK activity, indicating that PITALRE is an essential component
of TAK. PITALRE-containing complexes can hyperphosphorylate the CTD,
suggesting that PITALRE is the CTD kinase present in the TAK
complex. Additionally, we have observed that TAK can be activated
in peripheral blood lymphocytes and promonocytic cell lines, suggesting
that regulation of TAK catalytic function is one mechanism underlying
the sensitivity of HIV to its cellular environment (41). It
is notable that PITALRE is expressed at high levels in lymphoid tissue
relative to other human tissues (8).
To determine the role of TAK in Tat-mediated viral transcription, in
this study, wild-type PITALRE or catalytically inactive mutant PITALRE
was transiently overexpressed and the resultant effect on
transactivation of the HIV-1 long terminal repeat (LTR) by Tat-1 was
observed. Additionally, we determined whether PITALRE could activate
the transcription of the HIV-1 LTR when targeted directly to a
promoter-proximal RNA element. These studies have provided evidence
that TAK mediates Tat's transactivation function in vivo.
Transiently expressed wild-type PITALRE and catalytic mutant D167N
specifically associate with Tat in vitro.
Plasmid expression
vectors were constructed that express a FLAG epitope-tagged
wild-type or mutant PITALRE. The mutant protein, termed D167N, contains
a substitution of asparagine for aspartic acid at residue 167 which
inactivates catalytic function (12). To verify that the
plasmids express stable proteins and that the D167N mutation disrupts
catalytic function, HeLa cells were transfected with FLAG vector,
PITALRE, or D167N plasmid DNA. At 72 h posttransfection, lysates were prepared and immunoprecipitation was carried out with
anti-FLAG antibodies, followed by in vitro kinase assays (Fig.
1A). The results demonstrated that a
46-kDa autophosphorylated product was present in
PITALRE-transfected cells which was absent in D167N-transfected cells,
confirming disruption of PITALRE catalysis by the D167N substitution.
Immunoprecipitation of lysates with anti-FLAG antibodies followed by
immunoblotting with PITALRE antibodies demonstrated equivalent
expression levels for PITALRE and D167N (Fig. 1A).
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
PITALRE, the Catalytic Subunit of TAK, Is Required for Human
Immunodeficiency Virus Tat Transactivation In Vivo
ABSTRACT
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-D-ribofuranosylbenzimidazole (DRB)
(10, 38), (ii) DRB inhibits Tat-mediated transcription (27); (iii) DRB can specifically inhibit the activities of
some cellular kinases (44), and (iv) the
hyperphosphorylation of RNAPII on its repetitive carboxyl-terminal
domain (CTD) correlates with the transition from transcription
initiation to elongation (6, 29, 32). These results have
suggested that a DRB-sensitive CTD kinase is required for Tat function
and, further, that such a kinase could be the direct cofactor mediating
Tat function. Both TFIIH (43) and TAK (15) are
sensitive to DRB and are able to hyperphosphorylate the CTD of RNAPII.
Further, it has been observed that the CTD of RNAPII is required for
Tat function in cells (4, 30, 42) and that purified Tat is
able to enhance CTD phosphorylation in vitro by TFIIH and, perhaps,
other kinases (11, 31). Thus, it seems probable that a
Tat cofactor is a DRB-sensitive kinase(s) which can
phosphorylate RNAPII on its CTD.
View larger version (16K):
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FIG. 1.
Transiently expressed wild-type and catalytic mutant
PITALRE proteins specifically associate with Tat in vitro. HeLa cells
(30% confluent in 10-cm-diameter dishes) were transfected by
Lipofectamine (Life Tech) with 3 µg of a CMV2-FLAG or
CMV2-FLAG-PITALRE plasmid, and at 72 h posttransfection, 1-ml
lysates were prepared as previously described (42). (A)
Kinase assays and immunoblots. Lysates (500 µl) were
immunoprecipitated with 5 µl of anti-FLAG monoclonal antibody of M2
(3 mg/ml; Kodak). For kinase assays, immunoprecipitates were bound to
protein A-Sepharose (RepliGen), washed three times in EBC (50 mM
Tris-HCl [pH 8.0], 120 mM NaCl, 0.5% Nonidet P-40, 5 mM
dithiothreitol) plus 0.03% sodium dodecyl sulfate, and in vitro kinase
assays were performed as previously described (15); 50% of
the reactions were analyzed on a sodium dodecyl sulfate-9%
polyacrylamide gel. For immunoblots, lysates (500 µl) were
immunoprecipitated with 5 µl of anti-FLAG monoclonal antibody M2 and
bound and washed as described above, and 50% of the immunoprecipitates
were loaded onto a sodium dodecyl sulfate-8% polyacrylamide gel and
then transferred to a nitrocellulose membrane; to detect PITALRE
proteins, an enhanced-chemiluminescence procedure was used with a
commercial PITALRE antiserum (Santa Cruz Biotechnology). (B) GST-Tat-2
in vitro binding assay. Lysates (500 µl) were incubated with 0.5 µg
of GST-Tat2 or GST-C59A (Tat-2) attached to glutathionine-Sepharose
beads as described previously (15). After incubations, the
bead complexes were washed three times in EBC plus 0.03% sodium
dodecyl sulfate and then loaded onto a sodium dodecyl sulfate-8%
polyacrylamide gel. The gel was transferred to nitrocellulose and
probed with an anti-PITALRE antibody as described above. The values
beside the gels are molecular weights in thousands.
PITALRE overexpression in HeLa cells inhibits transactivation by
Tat-1.
To examine the effects of overexpression of wild-type and
catalytic mutant PITALRE proteins on Tat function in vivo, HeLa cells
were cotransfected with an HIV-1 LTR luciferase reporter plasmid, a
Tat-1 expression plasmid, and a plasmid expressing PITALRE or D167N
(Fig. 2A). Measurement of luciferase
activities 72 h posttransfection and normalization to
-galactosidase activities from an internal reference plasmid
indicated that Tat-1 activated reporter expression 55-fold in the
presence of the parental FLAG vector. PITALRE overexpression
resulted in 2.7-fold inhibition of Tat transactivation function,
and overexpression of D167N resulted in 5.8-fold inhibition of
transactivation. Overexpression of the wild-type and D167N proteins had
relatively little effect on basal-level expression of the HIV-1 LTR
luciferase plasmid; in several independent experiments, the wild-type
and D167N proteins reduced basal expression from 1.1-fold to 1.4-fold
and from 1.3-fold to 1.9-fold, respectively (also see Fig. 3).
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-galactosidase activities indicated 290-fold Tax
activation in the presence of the FLAG parent vector, 433-fold
activation upon PITALRE overexpression, and 1,260-fold activation upon
overexpression of D167N. In multiple separate experiments, D167N
reproducibly enhanced Tax transactivation 1.5- to 4.4-fold. The
significance of the increase in Tax transactivation by overexpression
of the D167N protein is unclear. However, our results imply that
PITALRE function may repress the expression of some promoters such as
the HTLV-1 LTR. Such a possibility has a precedent in the yeast SRB10
protein, which is a homolog of mammalian CDK8; SRB10 is a CTD kinase,
and genetic evidence indicates that it can function as both an
activator and a suppressor of transcription (19, 21, 24,
40).
Overexpression of the wild-type and D167N proteins did not
significantly affect basal-level expression of the HTLV-1 LTR (Fig. 2).
We conclude from these experiments that transactivation of the HTLV-1 LTR by Tax was not inhibited by PITALRE or
PITALRE-D167N expression, indicating that PITALRE overexpression does
not generally squelch activated transcription. It may be noteworthy
that Tax function does not require the CTD of RNAPII (4).
In the experiment presented in Fig. 2, the Tat, Tax, and PITALRE
proteins were expressed from the same cytomegalovirus (CMV) promoter;
therefore, it is highly unlikely that the levels of inhibition of Tat
transactivation by the wild-type and D167N PITALRE proteins can be
explained by reduced Tat protein levels. These results indicate that
both wild-type PITALRE and D167N, upon overexpression in HeLa cells,
can specifically squelch Tat-mediated transactivation. The ability of
wild-type overexpressed PITALRE to squelch Tat function suggests that
TAK function requires one or more subunits in addition to PITALRE, such
that overexpression of one subunit disrupts the stoichiometry of the
functional complex. The observation that D167N squelches Tat function
more efficiently than does wild-type PITALRE suggests that the
catalytic function of PITALRE is important for TAK function and is
consistent with our previously proposed model that the function of TAK
during HIV transcription is to phosphorylate the CTD of RNAPII
(15).
PITALRE overexpression inhibits transactivation of an integrated HIV-1 LTR. To determine whether PITALRE overexpression could inhibit Tat activation of an integrated HIV-1 LTR, we examined Jurkat T-cell line 1G5, which contains an integrated HIV-1 LTR with luciferase as the reporter protein (1). 1G5 cells were cotransfected by electroporation with a Tat-1 expression plasmid and a PITALRE or D167N expression plasmid (Fig. 3). Measurement of Tat-induced luciferase activities at 24 h posttransfection yielded 60.1 luciferase U in the presence of the parental vector, 18.4 U upon wild-type PITALRE overexpression, and 7.8 U upon D167N overexpression. No inhibition of basal luciferase units by PITALRE or D167N was observed. Therefore, in 1G5 cells, PITALRE overexpression inhibited Tat-induced luciferase expression 3.3-fold, while D167N overexpression inhibited Tat-induced luciferase expression 7.7-fold.
|
PITALRE fused to Rev activates an HIV-1 LTR containing an RRE in place of TAR. Our observation that Tat associates with TAK led us to suggest the model for the mechanism of action of Tat shown in Fig. 4A. This model proposes that by binding to TAR RNA, Tat is able to functionally recruit TAK to the RNAPII complex, leading to hyperphosphorylation of the CTD and a resultant activation of transcription (15). To test this model, we investigated whether gene expression directed by the HIV-1 LTR sequences can be activated in vivo by direct recruitment of PITALRE to a promoter-proximal RNA element. For these experiments, we utilized an HIV-1 LTR-chloramphenicol acetyltransferase (CAT) reporter plasmid containing the minimal HIV-1 Rev response element (RRE) in place of the TAR element (39). This reporter has been shown to be activated by fusions of the Rev protein to Tat or herpesvirus VP16, but not by the other transcriptional activators tested (23, 39).
|
RRE) was not
activated by PITALRE-Rev. In an additional experiment, we compared
activation of the reporter plasmid by PITALRE-Rev and Tat-Rev fusion
proteins (Fig. 5B). The Tat-Rev protein was considerably more active,
resulting in 37-fold activation versus 4.8-fold activation by the
PITALRE-Rev protein.
|
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ACKNOWLEDGMENTS |
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We gratefully acknowledge Susan Marriott, Mark Goff, Bryan Cullen, and John Belmont for useful reagents.
This work was supported by NIH grant AI35381 and Training Program T32 AI07483 for Basic Research and AIDS (M.O.G.).
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ADDENDUM IN PROOF |
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The cyclin partner of PITALRE has recently been identified (P. Wei, M. E. Garber, S. M. Fang, W. H. Fischer, and K. A. Jones, Cell 92:451-462, 1998). PITALRE has consequently been termed CDK9 and its cyclin partner has been termed cyclin T.
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Molecular Virology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Phone: (713) 798-5774. Fax: (713) 798-3490. E-mail: arice{at}bcm.tmc.edu.
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